The central role of heme-copper oxidases in biological energy production has made them the targets of intense study in many laboratories across the globe. Since the crystal structures have now defined the coordination of the redox centers and the identity of proton pumping channels, attention has focused on determining mechanistic details. Unraveling these mechanisms has required application of specialized spectroscopic and biophysical techniques. Our contribution has been the application of X-ray absorption spectroscopy (XAS) to uniquely define the coordination of the copper centers in a number of derivatives of different members of the oxidase family. The lack of optical or magnetic signals from CuB makes XAS particularly relevant to testing proposed structures for resting and intermediate states of the binuclear center. In this renewal application, we will extend our studies to include novel members of the oxidase family and catalytic intermediates. First, we will investigate resting states of the CuB centers of quinol oxidases from Bacillus subtilis, Acidianus ambivalens, Thioalcalomicrobium aerophilum, and a CuA-deleted mutant of Thermus thermophilus. The reduced forms will be examined carefully for evidence of functionally important histidine dissociation and chloride ligation as recently observed for the b(03) enzyme. We will next determine the structures of key intermediates in the reaction pathway, again searching for evidence of histidine dissociation from CuB proposed to be an important element of proton pumping. These data will be backed up by studies on inorganic and engineered protein models for cytochrome oxidase. Finally, we will begin to explore the mechanism of interaction of cytochrome oxidase with recently discovered metallochaperones, which are believed to be essential for proper maturation of the intact oxidase in the membrane.